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영어

Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. In this study, we designed the insect cell expression system to produce anti-cancer mAb CO17-1A, which recognizes the antigen GA733 highly expressed on the surface membrane of colorectal carcinoma cells. The heavy (HC) and light chain (LC) genes of the mAb CO17-1A were cloned under the control of two different promoters, P10 and PPH, respectively on baculovirus expression, pFastBacTM Dual vector. The gene expression cassettes carrying HC and LC genes were transfered into a parent bacmid in E.coli (DH10Bac). The bacmid was transfected to Sf9 insect cells to generate the baculovirus expressing mAb CO17-1A (CO17-1A-Bac virus). Western blot and immunoflouorescence confocal analyses confirmed the mAb CO17-1A expression in CO17-1A-Bac virus infected insect cells. The optimum conditions for mAb expression were validated at 24, 48 and 72h after the virus infection with MOI (optimum virus concentration) ranging (0.2, 1 and 5). Expression of mAb CO17-1A in insect cells significantly increased at both 48 and 72h after transfection with the MOI 1. HPLC chromatography revealed that the mAb CO17-1A expressed in the insect cell had insect specific glycan structures. Cell ELISA showed that the purified mAb from insect cell cultured media had a specific binding activity to SW948 human colorectal cancer cell. These results indicated that the baculovirus insect cell system is able to express, assemble, and secrete functional full size monoclonal antibody with insect specific glycosylation.